0270~6474/83/0306-1279$02.00/O The Journal of Neuroscience Copyright 0 Society for Neuroscience Vol. 3,, No. 6, pp. 1279-1288 Printed in U.S.A. June 1983 FAST AXONAL TRANSPORT IN PERMEABILIZED LOBSTER GIANT AXONS IS INHIBITED BY VANADATE’ DAVID S. FORMAN,*, 2 KURT J. BROWN,* AND DAVID R. LIVENGOODS * Department of Anatomy, Uniformed Services University of the Health Sciences and $ Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20814 Received October 22, 1982; Revised January 17, 1983; Accepted January 18, 1983 Abstract We have developed a method for permeabilizing axons and reactivating the fast transport of microscopically visible organelles. Saltatory movements of organelles in motor axons isolated from lobster walking legs were observed using Nomarski optics and time-lapse video microscopy. In the center of the axon most of the particles and mitochondria moved in the retrograde direction, but immediately below the axolemma the majority moved in the anterograde direction. When axons were permeabilized with 0.02% saponin in an adenosine 5’-triphosphate (ATP)-free “internal” medium, all organelle movement ceased. Saltatory movements resembling those in intact axons immediately reappeared upon the addition of MgATP. Very slight movement could be detected with ATP concentrations as low as 10 PM, and movement appeared to be maximal with 1 to 5 mM ATP. Vanadate, which does not affect axonal transport in intact axons, inhibited the reactivated organelle movements in permeabilized axons. Movement was rapidly and reversibly inhibited by 50 to 100 PM sodium orthovanadate. The effects of vanadate, including the time course of inhibition, its reversibility, and its concentration dependence, are consistent with the hypothesis that a dynein- like molecule may play a role in the mechanism of fast axonal transport. Fast axonal transport can be directly visualized in the mechanism of transport do not cross the axonal living axons by light microscopy (Smith, 1971,1977,1980; plasma membrane. One approach to circumvent this Kirkpatrick et al., 1972; Cooper and Smith, 1974; Forman problem that has been useful for studying other types of et al., 1977a, b; Forman and Shain, 1981; Allen et al., cell motility is to destroy the permeability barrier of the 1982a, b; Forman, 1982c; reviewed in Grafstein and For- plasma membrane with detergents in a medium that man, 1980). Microscopically visible organelles move in permits reactivation of the movement. Detergent-per- both the anterograde and retrograde directions by salta- meabilized cells have proved useful for analyzing the tory movements similar to those seen in a variety of mechanisms underlying other types of cell motility, such other cells (Rebhun, 1972; Forman, 1982b). The cellular as muscle contraction, ciliary motion, chromosome move- mechanism of fast transport is poorly understood. Many ment, and cytokinesis (Cande and Wolniak, 1978; Cande experimental probes that might be useful for studying et al., 1981). Recently, methods have been reported for reactivating saltatory movement in fibroblasts (Forman, 1981; 1982b), chromatophores (Clark and Rosenbaum, ’ This work was supported by the Uniformed Services University of 1982; Stearns and Ochs, 1982) and neuroblastoma cells the Health Sciences, Protocols R07035 and R07032, and United States (Stearns and Boggs, 1981) permeabilized with detergent, Public Health Service Grant GM30450. The opinions or assertions and in isolated squid axoplasm (Brady et al., 1982). In contained herein are the private ones of the authors and are not to be this paper we describe a method for permeabilizing lob- construed as official or reflecting the views of the Department of ster axons with saponin and reactivating fast transport Defense or the Uniformed Services University of the Health Sciences. with exogenous ATP. We have used this method to study We greatly appreciate a loan of equipment from the Naval Medical the effects of vanadate, an inhibitor of dynein (the ATP- Research Institute, Bethesda, Maryland. We wish to thank Mark ase involved in the movement of cilia and flagella, re- Promersberger for excellent technical assistance, Dr. Mark Adelman viewed in Warner and Mitchell, 1980). Vanadate, which for valuable discussion, and Clayton Cisar and Ed Lundgren, Labora- cannot normally enter living axons, reversibly inhibits tory Animal Medicine Department, Uniformed Services University of the Health Sciences, for help in procuring lobsters. fast organelle transport in the permeabilized axons. This ‘To whom correspondence should be addressed at Department of finding suggests that a cytoplasmic dynein-like molecule Anatomy, Uniformed Services University of the Health Sciences, may play a role in the mechanism of fast axonal trans- School of Medicine, 4301 Jones Bridge Road, Bethesda, MD 20814. port. 1279 1280 Forman et al. Vol. 3, No. 6, June 1983 Materials and Methods was covered by a 45 x 45 mm mask (equivalent to an Isolation of giant axons. Lobsters (Homarus ameri- area of 100 mm’). Long saltations and “tugging move- canus) from commercial sources, weighing 0.5 to 1 kg, ments” (see “Results”) with net displacements of greater were maintained in aerated holding tanks in artificial sea than 1 pm were counted during successive 1-min periods water at 4°C for up to 3 days. Walking legs were removed of videotape record. Counts from four equally spaced immediately before dissection and placed in an regions (totaling 40% of the screen area) were added “external” medium containing 450 mM NaCl, 15 mu KCl, together to give a representative index of the frequency 25 mu CaCL, 4 mM MgS04, 4 mu MgC12, and 10 mu of organelle movements (see Forman, 1982a). Particle HEPES (sodium salt), pH 7.4. Motor nerves in the mer- velocities were measured from videotape records using a opodite (Wiersma, 1961) were exposed, and the proximal 610E Video Pointer (Colorado Video, Boulder, Colorado). and distal ends of 3- to 5-cm-long segments were tied Permeabilization. Organelle movements inside an with surgical threads of different diameters so that the axon were observed in external medium to provide a base direction of movement could later be identified. The line. The axon was then incubated for 10 min in an nerves were placed in 85-mm plastic Petri dishes contain- “internal” medium (modified from Abercrombie et al., ing about 15 ml of external medium and were anchored 1981) that contained 300 mM potassium aspartate, 20 mM by pushing the suture ends into a 5-mm-thick layer of sodium aspartate, 344 mM glycine, 10 mM MgCL, 5 mu Sylgard (Dow Corning) on the bottom. Single giant motor EGTA, and 10 mM HEPES, pH 7.3. The axon was then axons or adherent pairs were isolated using a Zeiss mi- permeabilized by incubation for 15 min in internal me- crosurgical dissecting microscope and darkfield illumi- dium plus 0.02% saponin (Sigma) and 1% dimethyl sulf- nation. In most experiments the axons studied were oxide (DMSO). The saponin was added to the medium either the “fast closer” or one of the two “opener” axons from a stock solution of 2% (w/v) saponin dissolved in to the dactylopodite (Wiersma, 1961; Kravitz et al., 1965); DMSO. DMSO alone (1%) did not permeabilize the axon there was no obvious difference in movement between or noticeably affect transport. The axon was then per- the different types of axons. The diameters of the axons fused with various reactivation media consisting of ad- ranged from 30 to 50 pm. ditional components (described under “Results”) dis- A coverslip longer than the nerve segment was slipped solved in internal medium without saponin or DMSO. underneath it, and the medium was gently removed with Results a syringe. The coverslip with the adhering axon was then quickly inverted and lowered onto a pool of medium on Fast axonal transport of microscopically visible or- a spacer slide. The spacer slide consisted of a 74 mm x ganelles in intact axons. With conventional Nomarski 50 mm glass slide to which two 60-mm long no. 1 cover- optics, numerous organelles can be seen moving inside slips were glued with Permabond 910 adhesive, leaving a living lobster giant axons. The sheath surrounding each gap of 10 mm between them. The sides of the coverslip isolated axon, consisting of several layers of thin glial were sealed with melted Kronig wax. Solutions were cells and collagen fibers, did not excessively interfere changed by pipetting fluid at one end of the coverslip and with good visualization of organelles in the axoplasm absorbing it at the other end with filter paper. (Fig. 1). The organelles and their movements resembled Video microscopy. The axons were observed with No- those that have been described in other types of axons marski differential interference contrast optics using a (Smith, 1971, 1972, 1977, 1980; Kirkpatrick et al., 1972; Zeiss basic microscope with a X 40 planachromatic ob- Cooper and Smith, 1974; Forman et al., 1977a, b; reviewed jective (numerical aperture = 0.65). A Calflex heat re- in Grafstein and Forman, 1980; Forman, 1982b). Briefly, flection filter was used to protect the specimen, and a two classes of organelles could be distinguished: elon- Zeiss VG9 filter provided monochromatic (546 & 5 nm) gated mitochondria and small, round or ellipsoidal, vesic- light. Best results were obtained with the polarizer par- ular “particles” (Figs. 1 and 2, a to c).~ Mitochondria allel to the axon. The microscopic image was magnified varied in length from a few micrometers to longer than and recorded using a video microscopy system (Wil- 40 w and were usually aligned parallel to the long axis lingham and Pastan, 1978) consisting of a highly sensitive of the axon. Mitochondria were found distributed SIT video camera (RCA TC1030H), a time-lapse video- throughout the axoplasm but were especially concen- tape recorder (Panasonic NV-8030 or NV-8050), and a trated immediately below the axonal plasma membrane. monitor (RCA TC1209). Time was continuously dis- More particles than mitochondria were seen moving played on the video record by a date and time generator (Table I).